Optogenetics refers to techniques involving the use of genetically encoded proteins, which fluoresce or change conformation upon absorption of visible light, as reporters or actuators respectively, of biological systems (Knöpfel et al., 2010; Alford et al., 2013). The prototypical optogenetic reporter is the Aequorea victoria green FP (GFP), while the prototypical optogenetic actuator is channelrhodopsin-2 (Chr2) from the algae Chlamydomonas reinhardtii (Boyden et al., 2005). GFP is a genetically encoded fluorophore that enables visualization of the localization and dynamics of chimeric fusion proteins in live cells (Tsien, 1998). ChR2 is a light activated channel that opens upon illumination, allowing cations to enter the cell which in turn causes a depolarization of the membrane potential (Nagel et al., 2003; Boyden et al., 2005; Fenno et al., 2011; Yizhar et al., 2011). These examples have inspired the development of variations to expand the range of optogenetic applications (Campbell and Davidson, 2010; Ibraheem and Campbell, 2010; Zhao et al., 2011; Mutoh et al., 2012; Jin et al., 2012; Chen et al., 2013; Hertel and Zhang, 2013; Wu et al., 2013).
For optogenetic actuators, there is a currently a limited toolbox of variants (Akerboom et al., 2013; Müller and Weber, 2013). There are several classes of optogenetic actuators. Opsin-based actuators are membrane-spanning channels that open and pass small ions in response to optical activation (Boyden et al., 2005; Zhang et al., 2007; Klare et al., 2008; Zhang et al., 2008; Airan et al., 2009; Berndt et al., 2009; Chow et al., 2010; Gunaydin et al., 2010; Knöpfel et al., 2010; Berndt et al., 2011; Fenno et al., 2011; Yizhar et al., 2011; Prigge et al., 2012; Karunarathne et al., 2013).
Allosteric-based actuators including BLUF, LOV, and PYP domains are typically small proteins (less than 140 amino acids) with a flavin adenine dinucleotide or flavin mononucleotide chromophore cofactor. Blue light illumination causes structural changes that unfold the C-terminal α-helix (Iseki et al., 2002; Schröder-Lang et al., 2007; Stierl et al., 2011; Christie et al., 2012). Attempts have been made to exploit the conformational change in these domains to modulate a desired genetically fused enzyme. Many of these efforts were inspired by the engineering of a photoactivatable hybrid between the GTPase Rac1 and a LOV domain (PA-Rac) (Wu et al., 2009). As Rac1 is involved in the regulation of cytoskeleton remodeling, localized illumination of a cell expressing PA-Rac stimulates cell migration in an experimentally controllable way. Replicating the success of PA-Rac with other enzymes has been challenging (Mills et al., 2011; Schierling and Pingoud, 2012). In the case of the LOV-luciferase hybrid, only about a 20% decrease in activity was ultimately achieved upon illumination (Hattori et al., 2013). Efforts have been made to use LOV domains for purposes other than enzyme control including caging of binding peptides (Lungu et al., 2012) and photo control of a protein degradation sequence (Renicke et al., 2013). Attempts to make such constructs have failed and provide only modest light dependent modulation (Strickland et al., 2010).
Oligomerization-based actuators are proteins which undergo light-dependent modulation of quaternary structure (Yazawa et al., 2009; Strickland et al., 2012; Zhou et al., 2012). Oligomerization-based actuators are the most diverse class of actuators, but also the one with the greatest redundancy in terms of functionality, and undergo a change in intermolecular interactions (i.e., formation or dissociation of homo- or heterodimers or higher order oligomers) upon illumination (Shimizu-Sato et al., 2002; Levskaya et al., 2009; Yazawa et al., 2009; Kennedy et al., 2010; Toettcher et al., 2011; Christie et al., 2012; Idevall-Hagren et al., 2012; Strickland et al., 2012; Wu et al., 2012; Zhou et al., 2012; Bugaj et al., 2013; Kakumoto and Nakata, 2013; Pathak et al., 2013; Yang et al., 2013). As optogenetic actuators, light-activated oligomerizers are generally restricted to being applied for either reconstitution of split proteins or perturbation of protein subcellular localization.
An ideal optogenetic tool is one in which all components, including the chromophore, are proteinaceous. The prior art actuators described above require chromophore cofactors that are normally present in cells or can be introduced into cells either by incubation in solution (Levskaya et al., 2009) or introduction of the required biosynthetic genes (Müller et al., 2013). This requirement for chromophore cofactors is not problematic in many in vitro applications, but for in vivo applications the accompanying depletion of cellular cofactors or the need for systematic delivery presents complications. Oligomerization-based actuators that are fully proteinaceous avoid these problems and are better suited to in vivo applications. A fully proteinaceous actuator, UVR8, uses its intrinsic tryptophan residues to absorb UV light and has been exploited as both a light dissociable homodimer and a light induced heterodimer with COP1 (Rizzini et al., 2011; Crefcoeur et al., 2013). One recent application of the UVR8-COP1 system was the photocontrol of protein secretion by dissociation of oligomerized vesicular stomatitis virus glycoprotein in dissociated neurons (Chen et al., 2013). However, the UVR8 system requires high energy UV light, making it unsuitable for in vivo applications. The Dronpa FP-based reversible tetramer dissociation system appears promising (Zhou et al., 2012). As a homologue of Aequorea GFP, Dronpa autogenically forms a visible wavelength chromophore within the protected interior of its β-barrel structure. A light-induced change in the conformation of the chromophore triggers structural changes in the β-barrel that lead to dissociation of the Dronpa tetramer (Mizuno et al., 2008). This approach has been used for reversible release from the plasma membrane and caging of proteins due to a combination of clustering, structural perturbation, and active site obstruction. Implementing the Dronpa-based caging of enzymes is challenging since successful designs are largely based on trial and error, and it is difficult to fully turn proteins off in the dark or oligomerized state.
Thus, there exists a need in the art for the continued development of fluorescent proteins for use in scientific applications which may mitigate the technical limitations of the prior art.